U.S. patent number 8,999,176 [Application Number 14/145,087] was granted by the patent office on 2015-04-07 for isolation of nucleic acids.
This patent grant is currently assigned to Exact Sciences Corporation. The grantee listed for this patent is Exact Sciences Corporation. Invention is credited to Michael J. Domanico, Keith Kopitzke, James P. Light, II, John Zeis.
United States Patent |
8,999,176 |
Domanico , et al. |
April 7, 2015 |
Isolation of nucleic acids
Abstract
Provided herein is technology relating to isolating nucleic
acids. In particular, the technology relates to methods and kits
for extracting nucleic acids from problematic samples such as
stool.
Inventors: |
Domanico; Michael J.
(Middleton, WI), Light, II; James P. (Middleton, WI),
Kopitzke; Keith (Fallbrook, CA), Zeis; John (San Marcos,
CA) |
Applicant: |
Name |
City |
State |
Country |
Type |
Exact Sciences Corporation |
Madison |
WI |
US |
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Assignee: |
Exact Sciences Corporation
(Madison, WI)
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Family
ID: |
47140037 |
Appl.
No.: |
14/145,087 |
Filed: |
December 31, 2013 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20140194609 A1 |
Jul 10, 2014 |
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Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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PCT/US2012/037581 |
May 11, 2012 |
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61485338 |
May 12, 2011 |
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61485386 |
May 12, 2011 |
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61485448 |
May 12, 2011 |
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61485214 |
May 12, 2011 |
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Current U.S.
Class: |
210/781 |
Current CPC
Class: |
B01D
33/15 (20130101); B01L 3/5021 (20130101); C12Q
1/6806 (20130101); B01L 3/00 (20130101); C12N
15/1006 (20130101); B01D 33/155 (20130101); B03C
1/30 (20130101); C12N 15/1013 (20130101); C12N
15/1017 (20130101); B04B 3/00 (20130101); C12Q
1/6886 (20130101); C12Q 2600/158 (20130101); C12Q
2600/16 (20130101); Y10T 436/143333 (20150115) |
Current International
Class: |
C12Q
1/68 (20060101); B01L 3/00 (20060101); C12N
15/10 (20060101); B04B 3/00 (20060101); B03C
1/30 (20060101); B01D 33/15 (20060101) |
Field of
Search: |
;210/781,806,297,360.1,380.1 ;435/297.3 ;422/72 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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265244 |
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Sep 1992 |
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EP |
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90/11345 |
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Oct 1990 |
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WO |
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2004108925 |
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Dec 2004 |
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WO |
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2005023091 |
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Mar 2005 |
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WO |
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2008150826 |
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Dec 2008 |
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WO |
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2010014970 |
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Feb 2010 |
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WO |
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2010028382 |
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Mar 2010 |
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WO |
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2011014970 |
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Feb 2011 |
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WO |
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2012002887 |
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Jan 2012 |
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WO |
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WO 2012155072 |
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Nov 2012 |
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WO |
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Other References
Ahlquist et al., "Colorectal Cancer Screening by Detection of
Altered Human DNA in Stool: Feasibility of a Multitarget Assay
Panel," Gastroenterology, 2000, 119: 1219-1227. cited by applicant
.
Berthelet et al., "Rapid, direct extraction of DNA from soils for
PCR analysis using polyvinylpolypyrrolidone spin columns," FEMS
Microbiology Letters, 1996, 138:17-22. cited by applicant .
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Environmental Sciences, University of Toledo, "Polyvinylpyrrolidone
(PVPP) cleanup of DNA samples," Dec. 2004, 2 pages. cited by
applicant .
Mangiapan et al. "Sequence Capture-PCR Improves Detection of
Mycobacterial DNA in Clinical Specimens," Journal of Clinical
Microbiology, 1996, 34. p. 1209-1215. cited by applicant .
Parham et al., "Specific Magnetic Bead-Based Capture of Genomic DNA
from Clinical Samples: Application to the Detection of Group B
Streptococci in Vaginal/Anal Swabs," Clinical Chemistry, 2007,
53:9, p. 1570-1576. cited by applicant .
"PVP in Stool Samples," MadSci Network: Molecular Biology, Nov. 20,
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Qiagen, QIAamp DNA Stool Mini Kit Handbook, Aug. 2001, 40 pages.
cited by applicant .
QIAamp.RTM. genomic DNA Kits, product information, Apr. 2008, 12
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in Stool Samples by a Reverse Line Hybridization Assay," J Clin
Microbiol., 2003, 41(11):5041-5045. cited by applicant .
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brief retrospective," Biotechniques, 2008, 44:701-704. cited by
applicant .
Whitney et al., "Enhanced Retrieval of DNA from Human Fecal Samples
Results in Improved Performance of Colorectal Cancer Screening
Test," JMD, 2004, 6(4). cited by applicant .
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Species," Clinical Microbiology Reviews, 2007, 20(3):511-532. cited
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Aquamira Technologies, "Some Important Words in Regards to Filter
Ratings," retrived Oct. 3, 2014. cited by applicant .
Cullen et al., "Simple and rapid method for direct extraction of
microbila DNA from soil for PCR", Soil Biology and Biochemistry,
vol. 30, No. 8/9, 1998, pp. 983-993. cited by applicant .
Doulton USA, "Absolute Vs. Nominal Microns Pore Ratings," Retrieved
Oct. 3, 2014, from http;//doultonusa.com/HTML
pages/absolute.sub.--vs.sub.--nominal.sub.--microns.sub.--rating.htm.
cited by applicant .
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No. 12782489.4, 9 pages. cited by applicant .
Lenntech B.V., "Absolute rating vs. nominal rating for filters,"
www.lenntech.com/library/fine/absolute/absolute-nominal-filters.htm,
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applicant.
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Primary Examiner: Reifsnyder; David A
Attorney, Agent or Firm: Casimir Jones, S.C.
Parent Case Text
The present application claims the benefit of U.S. Provisional
Patent Application Ser. Nos. 61/485,214, 61/485,338, 61/485,386,
and 61/485,448, each of which was filed May 12, 2011, and each of
which is incorporated herein by reference in its entirety.
Claims
We claim:
1. A method of producing a filtrate from a sample, the method
comprising: a) placing a sample to be filtered into a spin filter,
said spin filter comprising i) a hollow body (1); ii) a bottom end
(2); and iii) an opening (3) at a top end of said hollow body (1)
opposite the bottom end (2), wherein said hollow body (1) and said
bottom end (2) are both composed of the same porous filtering
material; and b) centrifuging said spin filter, wherein during said
centrifuging, a fraction of said sample passes through said porous
filtering material of said spin filter to produce a filtrate.
2. The method of claim 1 wherein the porous filtering material of
said hollow body (1) and said bottom end (2) of said spin filter is
polyethylene.
3. The method of claim 1 wherein the porous filtering material of
said hollow body (1) and said bottom end (2) of said spin filter
has a nominal pore size of 20 micrometers.
4. The method of claim 1 wherein the bottom end (2) of said spin
filter has a shape selected from the group consisting of a
hemisphere, a disc, a cone, or a portion of an ellipsoid.
5. A method of claim 1, wherein said spin filter is placed in a
collection vessel adapted to receive the spin filter, and wherein
during said centrifuging, said filtrate is retained in said
collection vessel.
Description
FIELD OF INVENTION
Provided herein is technology relating to isolating nucleic acids.
In particular, the technology relates to methods and kits for
extracting nucleic acids from problematic samples such as
stool.
BACKGROUND
Isolating specific target nucleic acids from a sample is an
important step for many medical diagnostic assays. For example,
certain mutations and methylation states in known genes are
correlated, associated, and/or predictive of disease. DNA harboring
these genes can be recovered from a sample and tested for the
presence of the particular mutations and methylation states.
In practice, such assays require isolating and assaying several
genetic targets from a sample. For many detection methods,
detecting rare mutations or methylation events in a single gene
requires isolating and testing a large quantity of DNA. This
problem is compounded when assaying a panel of genes, each of which
must be present in a large quantity for a robust diagnostic test.
Thus, to detect rare mutations and methylation events in multiple
genes, the isolated DNA must be highly concentrated and comprise a
substantial portion of the detection assay.
This requirement imposes many problems, however. For example,
preparing such quantities and concentrations of DNA requires a
large sample as input (e.g., having a mass of several grams, e.g.,
approximately 2-4 grams) to provide sufficient nucleic acid for
detection, and thus requires a method that can prepare DNA from a
large sample. In addition, assay inhibitors are often isolated and
concentrated with the DNA preparation. Consequently, concentrated
DNA preparations produced by conventional methods also often retain
unacceptable concentrations of inhibitors, which are then
introduced into a subsequent assay. Moreover, if all targets of the
panel are extracted simultaneously in a bulk, non-selective DNA
preparation, the sensitivity of the assay is compromised because,
as the preparation is divided into aliquots for testing, less
extracted DNA from any one gene of the panel is present in the
assay. If, on the other hand, all members of the panel are
extracted and tested together and are thus present in the same
assay mixture, the sensitivity of detecting any single particular
target is compromised by the presence of the non-target DNA
molecules.
In addition, if a particular diagnostic target is present in a
complex sample, it will be present in a small amount relative to
other materials--both nucleic acid and non-nucleic acid--in the
sample, thus providing a challenge for analytical methods designed
to detect it. For example, analyses of DNA from stool samples is
complicated by the fact that bacteria compose approximately 60% of
the dry mass of feces and the remainder is largely the remains of
plant and animal matter ingested as food by the subject. As such,
the human subject's cells, which are only those that slough off the
lining of the digestive tract, are a very small fraction of the
stool and substantial amounts of nucleic acids from other sources
are present. Furthermore, in assays to detect gene modifications
indicative of colon cancer, cells derived from a tumor that may be
present in the colon would compose only a small fraction of the
human subject's gut cells that slough off the digestive tract
lining. Consequently, cancer cells (and the DNAs they contain) make
up a minimal amount of the stool mass. Such samples are also often
very viscous, which presents problems in sample preparation and
isolation of nucleic acid.
Conventional methods and kits for isolating DNA from samples
typically prepare total DNA (e.g., by a non-specific precipitation
method) from a sample. For complex samples such as stool samples,
this is a particular drawback of conventional methods, as total DNA
isolated from a stool sample comprises DNA from the gut-resident
bacteria (and any viruses, eukaryotes, and archaea present) along
with DNA from the subject. Moreover, conventional methods and kits
are primarily designed to prepare DNA from small samples, e.g.,
samples having masses of less than 1 gram, e.g., 50 to 200
milligrams, limiting the yield of target nucleic acid from complex
samples to very small amounts. Additional drawbacks are that most
conventional technology does not effectively remove inhibitors and
often require long processing steps, e.g., incubations.
Consequently, conventional methods are not suited to
high-sensitivity and high-specificity multi-gene panel analysis
because they cannot prepare sufficient amounts of highly
concentrated, inhibitor-free DNA from large samples, such as a
stool sample of several grams. Assays using DNA prepared with
conventional methods will not provide a sample that can be assayed
with the required sensitivity threshold for detecting rare mutation
or methylation events. Using a conventional method or kit to attain
the starting quantities needed to attain such sensitivity requires
multiple DNA extractions (e.g., the use of multiple kits) from
multiple samples in addition to extra purification steps to remove
inhibitors. Therefore, what is needed is a method of preparing
concentrated, inhibitor-free DNA from a sample for each member of a
gene panel for use in diagnostic assays.
SUMMARY
Provided herein is technology relating to isolating nucleic acids.
In particular, the technology relates to methods, systems, and kits
for extracting and purifying nucleic acids from exfoliated
intestinal cells in stool specimens for use in quantitative and
sensitive assays. The technology is embodied in a novel method for
purifying specific DNA from stool that utilizes inhibitor removal
steps and direct capture of DNA from stool supernatant, or a
combination of these steps. The technology further provides
filtration devices suitable for use with complex and viscous
samples, such as stool samples. Accordingly, provided herein is a
method for isolating a target nucleic acid from a sample, the
method comprising removing an assay inhibitor, if present, from the
sample to produce a clarified sample; capturing the target nucleic
acid, if present, from the clarified sample with a capture reagent
to form a capture complex; isolating the capture complex from the
clarified sample; and recovering the target nucleic acid, if
present, from the capture complex in a nucleic acid solution. In
some embodiments the method further comprises retaining the
clarified sample after the capturing step; and repeating the
isolating and recovering steps using the retained clarified sample
and a second capture reagent.
In some embodiments, removing the inhibitor comprises homogenizing
the sample to produce a homogenate; centrifuging the homogenate to
produce a supernatant; treating the supernatant with an
inhibitor-adsorbing composition to bind the inhibitor, if present,
in an inhibitor complex; and isolating the inhibitor complex from
the supernatant to produce a clarified sample. The
inhibitor-adsorbing composition in some embodiments is a
polyvinylpyrrolidone. In some embodiments, the polyvinylpyrrolidone
is insoluble and in some embodiments the polyvinylpyrrolidone is a
polyvinylpolypyrrolidone. It is useful in some embodiments to
provide the polyvinylpyrrolidone in a premeasured form, for example
in some embodiments the polyvinylpyrrolidone is provided as a
tablet. Various techniques are used to separate the inhibitor
complex from the sample. For example, in some embodiments isolating
the inhibitor complex comprises centrifuging to separate the
inhibitor complex from the supernatant.
In some embodiments, the centrifuging comprises centrifuging
through a spin column. Therefore, in some embodiments provided
herein is technology relating to filtration and particularly, but
not exclusively, to filters and methods for filtering by means of
centrifugation. Specifically, some embodiments of the technology
provided herein address the problem of spin filter clogging by
providing technology in which both the bottom end and body of a
spin filter are made from a porous or permeable material. That is,
the walls of the spin filter are made of the same or similar
material as that used for the filter means at the bottom end in
conventional designs. As such, when the bottom portion of the
filter becomes clogged during filtration, the walls provide
additional surface through which the sample can be filtered.
This technology is provided herein as a spin filter comprising a
hollow body, a bottom end, and an open top end opposite the bottom
end, wherein the hollow body is made from a porous filtering
material. In some embodiments the bottom end is made from a porous
filtering material. The hollow body and bottom end of the spin
filter assume any shape appropriate for the filtration application
to which the filter is applied. For example, in some embodiments
the hollow body is a tube and in some embodiments the bottom end is
a hemisphere. In other embodiments, the bottom end is a disc, a
cone, or a portion of an ellipsoid. Furthermore, the spin filter is
made from any material that is appropriate for filtering a sample.
Thus, in some embodiments the porous filtering material is
polyethylene. Samples comprise varying sizes of particles, matter,
precipitates, etc. that are to be removed by filtration.
Accordingly, the filtering material can be selected to have
physical properties that provide the desired separation. For
example, in some embodiments the porous filtering material has a
nominal pore size of 20 micrometers. In some embodiments, use of
the filter produces a filtrate that a user retains for additional
processing. As such, some embodiments provide a spin filter
assembly comprising a spin filter as described and a collection
vessel adapted to receive the spin filter and collect the
filtrate.
Also provided herein are methods for producing a filtrate from a
sample comprising placing a sample to be filtered into the spin
filter and centrifuging the spin filter, wherein during
centrifuging, a fraction of the sample passes through porous
filtering material of said spin filter to produce a filtrate.
The technology can be provided as a kit for use in a sample
separation. Embodiments of such a kit comprise a spin filter as
described and an instruction for use. In some embodiments the kit
further comprises a collection vessel. In some embodiments, a kit
comprising a spin filter further comprises additional reagents and
materials for sample preparation, e.g., for inhibitor removal
and/or target nucleic acid isolation.
In some embodiments, the methods and systems of the technology
comprise capturing a nucleic acid target. Capturing the target
nucleic acid, in some embodiments, comprises exposing a sample,
such as a clarified sample preparation, to a denaturing condition
to produce a denatured sample; and binding target nucleic acid in
the denatured sample to a capture reagent to form a capture
complex. Many treatments and conditions find use in denaturing
macromolecules such as DNA. For example, in some embodiments, the
denaturing condition comprises heating, e.g., in some embodiments
the denaturing condition comprises heating at 90.degree. C.
Supplementing the sample to be denatured facilitates the
denaturing; accordingly, in some embodiments, the clarified sample
further comprises a denaturant. In certain preferred embodiments,
the denaturant comprises guanidine thiocyanate. Furthermore, in
some embodiments the capture reagent comprises an oligonucleotide
complementary to at least a portion of the target nucleic acid. In
some preferred embodiments, the capture reagent comprises particle,
e.g., a magnetic particle. The oligonucleotide, in some embodiments
of the technology, hybridizes to at least a portion of the target
nucleic acid, and thus in some embodiments, the binding step
comprises hybridizing the oligonucleotide and the target nucleic
acid. Isolating the capture reagent (e.g., the capture
reagent/target nucleic acid complex) is accomplished in some
embodiments by exposing the capture reagent to a magnetic field;
that is, in some embodiments provided herein, the isolating step
comprises exposing the capture complex to a magnetic field and in
some embodiments exposing the capture complex to the magnetic field
localizes the target nucleic acid. The magnetic field is produced
by any appropriate magnet or magnetic device for the method. For
example, in some embodiments the isolating step comprises placing
the sample in a magnetic field produced by a first magnet oriented
with its north pole in close proximity to the sample and a second
magnet oriented with its south pole in close proximity to the
sample; and waiting for a time sufficient to allow the magnetic
field to move the magnetic particles to the desired location. A
device for producing a strong magnetic field is described, for
example, in U.S. patent application Ser. No. 13/089,116,
incorporated by reference herein.
The technologies provide for recovering target nucleic acid from
the capture reagent. In some embodiments, recovering the target
nucleic acid comprises eluting the target nucleic acid from the
capture complex, e.g., in some embodiments, by heating. In some
embodiments, elution of the target nucleic acid from the capture
complex comprises exposing the capture complex to high pH, e.g., in
some embodiments, by adding a solution of sodium hydroxide.
In some embodiments, the technology provides methods, systems and
kits for capturing multiple nucleic acids from a single sample,
e.g., a stool sample. For example, provided herein are methods for
isolating a nucleic acid from a stool sample comprising contacting
a stool sample with a target-specific capture reagent; binding a
target nucleic acid, when present, to the target-specific capture
reagent to form a complex; isolating the complex comprising the
target-specific capture reagent and the target nucleic acid, when
present, from the stool sample; eluting the target nucleic acid,
when present, from the complex to produce a target nucleic acid
solution comprising the target nucleic acid, when present; and
repeating the method using a different target-specific capture
reagent. The methods are appropriate for large samples, e.g.,
having a mass of at least 4 grams. Moreover, each eluted target
nucleic acid is sufficiently purified, sufficiently concentrated,
and sufficiently free of inhibitors such that each eluted target
nucleic acid, when present, is detected by a quantitative PCR when
the target nucleic acid solution composes up to approximately
one-third of a volume of the quantitative PCR.
In some embodiments of the methods provided, the target nucleic
acid is a human target nucleic acid. In additional embodiments, the
target nucleic acid is a DNA. While not limited in the means by
which the nucleic acid is isolated from the stool sample, in some
embodiments the target-specific capture reagent is a
sequence-specific nucleic acid capture reagent. In some
embodiments, the sequence-specific nucleic acid capture reagent is
an oligonucleotide and in some embodiments the oligonucleotide is
covalently attached to a magnetic or paramagnetic particle. Some
embodiments provide that a magnet is used for the isolating step
and some embodiments provide for the simultaneous isolation of more
than one target using multiple target-specific capture reagents in
a single isolation step.
The method is not limited in the types of samples that are
processed. For example, in some embodiments the sample is a viscous
sample, e.g., having a viscosity of more than ten centipoise in
some embodiments and having a viscosity of more than twenty
centipoise in some embodiments. Additionally, the samples are of a
wide range of sizes. The methods are used to process samples
having, in some embodiments, a mass of more than one gram and in
some embodiments the sample has a mass of more than five grams.
The technology provided herein is directed to removing inhibitors
from samples below an amount that inhibits an assay. Thus, in some
embodiments, the method provides that the nucleic acid solution
comprises a first amount of the assay inhibitor that is less than a
second amount of the assay inhibitor, wherein the second amount of
the assay inhibitor inhibits PCR when five microliters of the
nucleic acid solution are used in a PCR having a volume of
twenty-five microliters. In some embodiments, the nucleic acid
solution comprises a first amount of the assay inhibitor that is
less than a second amount of the assay inhibitor, wherein the
second amount of the assay inhibitor inhibits PCR when one
microliter of the nucleic acid solution is used in a PCR having a
volume of twenty-five microliters.
The technology is related to medical molecular diagnostics wherein
querying the state, presence, amount, sequence, etc., of a
biological substance (e.g., a molecule) is used to aid a medical
assessment. Accordingly, in some embodiments, the target nucleic
acid is correlated with a disease state selected from the set
consisting of colon cancer and adenoma.
The technology described herein is provided in a kit form in some
embodiments--for example, embodiments provide that the technology
is a kit for isolating a target nucleic acid from a sample
comprising a capture reagent comprising an oligonucleotide
covalently attached to a magnetic particle, an apparatus to produce
a magnetic field, polyvinylpyrrolidone, and an instruction for use.
In some embodiments, the kit further comprises a homogenization
solution. In some embodiments, the kit further comprises an elution
solution and in some embodiments the kit further comprises
guanidine thiocyanate. In some embodiments, it is convenient for
the polyvinylpyrrolidone to be in a premeasured form. For example,
the polyvinylpyrrolidone is provided in a tablet or capsule in some
embodiments. Some embodiments of the kit provide a spin filter for
removing polyvinylpyrrolidone.
In some embodiments, the target nucleic acid is isolated using a
magnetic field. As such, embodiments of the kits described herein
provide an apparatus that produces a magnetic field. One device
that is used to produce a magnetic field suitable for use with
embodiments of the technology provided herein comprises two magnets
or sets of magnets and places the north pole(s) of the first magnet
or set of magnets in close proximity to the sample and the south
pole(s) of the second magnet or set of magnets in close proximity
to the sample. In some embodiments, the kits further provide a
device for collecting a sample, e.g., a device having a body and a
detachable sample capsule attached to the body, wherein the
detachable sample capsule comprises a sample collection space
adapted to enclose a sample (for example, as described in U.S. Pat.
Appl. Ser. No. 61/476,707).
In some embodiments, the kit provides vessels (e.g., a tube, a
vial, a jar, and the like) used to process samples and hold various
compositions used to process samples or that result from processing
samples. For example, in some embodiments the kit further comprises
a vessel in which to hold the sample and in some embodiments the
kit further comprises a vessel in which to hold the isolated target
nucleic acid. The kit, in some embodiments, is used at a location
other than where the sample is processed and/or where the analyte
is assayed. Accordingly, in some embodiments the kit further
comprises a shipping container.
The technology provided herein finds use in systems for preparing a
nucleic acid from a sample. In some embodiments, the system
comprises polyvinylpyrrolidone for removing an inhibitor from the
sample, a reagent for capturing a target nucleic acid from the
sample, and a functionality for producing a magnetic field. In some
embodiments, the system further comprises a functionality for
collecting the sample and in some embodiments the system further
comprises a functionality for shipping the nucleic acid
solution.
Additional embodiments will be apparent to persons skilled in the
relevant art based on the teachings contained herein.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features, aspects, and advantages of the present
technology will become better understood with regard to the
following drawings:
FIGS. 1A and 1B provide charts of aspects of the nucleic acid
isolation process. FIG. 1A provides a chart showing the steps of
the nucleic acid isolation process. FIG. 1B is a flowchart showing
an embodiment of the process that finds use in the sequential
extraction of multiple targets from the same sample as an aspect of
the overall process of FIG. 1A.
FIG. 2 is a chemical structure of a polyvinylpyrrolidone.
FIG. 3 is a drawing of an exemplary spin filter.
FIG. 4 is a drawing showing an exploded view of the spin filter
shown in FIG. 3.
FIGS. 5A-5C are a series of drawings showing spin filter bottom
ends associated with the spin filter of FIGS. 3 and 4. FIG. 5A is a
drawing of a disc-shaped, solid (e.g., non-porous or non-permeable)
bottom end; FIG. 5B is a drawing of a disc-shaped, porous
(permeable) bottom end; FIG. 5C is a drawing of a porous, conical
bottom end.
FIG. 6 is a drawing of a spin filter assembled with a collection
tube.
FIG. 7 is a cut-away drawing of the spin filter depicted in FIG.
6.
FIGS. 8A and 8B are drawings of a spin filter comprising a body of
a porous material and a bottom end provided by a filter support.
FIG. 8A is an assembled view and FIG. 8B is an exploded view.
FIGS. 9A-9D are plots showing the removal of inhibitors from a
stool sample.
FIGS. 10A-10D are plots showing that spin filtration improves the
removal of inhibitors.
FIG. 11A is a plot of data comparing the localization efficiency of
the conventional technology for samples having viscosities of 1
centipoise and 25 centipoise. FIG. 11B is a plot of data comparing
the localization efficiency of the magnetic localization device
provided by Light and Miller (U.S. patent application Ser. No.
13/089,116) provided for samples having viscosities of 1 centipoise
and 25 centipoise.
FIG. 12A is a plot showing the results of a quantitative PCR in
which a single extraction from a stool sample recovers most of the
target DNA. FIG. 12B shows the concentrations of Gene A and Gene V
in nucleic acid solutions from a first extraction and a second
extraction.
FIGS. 13A-13D show plots showing the results of quantitative PCRs
in which the recoveries of four target DNAs are similar regardless
of the order in which the four target DNAs are extracted from a
stool sample.
FIG. 14 provides a chart comparing the workflow of an embodiment
(Process A) with an exemplary process for isolating DNA from stool
samples using steps based on existing methods (Process B, see,
e.g., WO 2010/028382).
DETAILED DESCRIPTION
The present technology is related to producing DNA samples and, in
particular, to methods for producing DNA samples that comprise
highly purified, low-abundance nucleic acids in a small volume
(e.g., less than 100, less than 60 microliters) and that are
substantially and/or effectively free of substances that inhibit
assays used to test the DNA samples (e.g., PCR, INVADER, QuARTS,
etc.). Such DNA samples find use in diagnostic assays that
qualitatively detect the presence of, or quantitatively measure the
activity, expression, or amount of, a gene, a gene variant (e.g.,
an allele), or a gene modification (e.g., methylation) present in a
sample taken from a patient. For example, some cancers are
correlated with the presence of particular mutant alleles or
particular methylation states, and thus detecting and/or
quantifying such mutant alleles or methylation states has
predictive value in the diagnosis and treatment of cancer.
Many valuable genetic markers are present in extremely low amounts
in samples and many of the events that produce such markers are
rare. Consequently, even sensitive detection methods such as PCR
require a large amount of DNA to provide enough of a low-abundance
target to meet or supersede the detection threshold of the assay.
Moreover, the presence of even low amounts of inhibitory substances
compromise the accuracy and precision of these assays directed to
detecting such low amounts of a target. Accordingly, provided
herein are methods providing the requisite management of volume and
concentration to produce such DNA samples.
Some biological samples, such as stool samples, contain a wide
variety of different compounds that are inhibitory to PCR. Thus,
the DNA extraction procedures include methods to remove and/or
inactivate PCR inhibitors. As such, provided herein is technology
relating to processing and preparing samples and particularly, but
not exclusively, to methods, systems, and kits for removing assay
inhibitors from samples comprising nucleic acids.
DEFINITIONS
To facilitate an understanding of the present technology, a number
of terms and phrases are defined below. Additional definitions are
set forth throughout the detailed description.
As used herein, "a" or "an" or "the" can mean one or more than one.
For example, "a" widget can mean one widget or a plurality of
widgets.
As used herein, an "inhibitor" means any compound, substance, or
composition, or combination thereof, that acts to decrease the
activity, precision, or accuracy of an assay, either directly or
indirectly, with respect to the activity, precision, or accuracy of
the assay when the inhibitor is absent. An inhibitor can be a
molecule, an atom, or a combination of molecules or atoms without
limitation.
As used herein, the process of passing a mixture through a filter
is called "filtration". The liquid produced after filtering a
suspension of a solid in a liquid is called "filtrate", while the
solid remaining in the filter is called "retentate", "residue", or
"filtrand".
As used herein, "insoluble" refers to the property that a substance
does not substantially dissolve in water and is essentially
immiscible therewith. Upon separation of an aqueous phase from a
non-aqueous phase, an insoluble substance does not partition into
or partition with the aqueous phase.
As used herein, the terms "subject" and "patient" refer to any
animal, such as a dog, cat, bird, livestock, and particularly a
mammal, preferably a human. In some instances, the subject is also
a "user" (and thus the user is also the subject or patient).
As used herein, the term "sample" and "specimen" are used
interchangeably, and in the broadest senses. In one sense, sample
is meant to include a specimen or culture obtained from any source,
as well as biological and environmental samples. Biological samples
may be obtained from animals (including humans) and encompass
fluids, solids, tissues, and gases. Biological samples include
blood products, such as plasma, serum, stool, urine, and the like.
Environmental samples include environmental material such as
surface matter, soil, mud, sludge, biofilms, water, crystals, and
industrial samples. Such examples are not however to be construed
as limiting the sample types applicable to the present
invention.
The term "target," when used in reference to a nucleic acid
capture, detection, or analysis method, generally refers to a
nucleic acid having a feature, e.g., a particular sequence of
nucleotides to be detected or analyzed, e.g., in a sample suspected
of containing the target nucleic acid. In some embodiments, a
target is a nucleic acid having a particular sequence for which it
is desirable to determine a methylation status. When used in
reference to the polymerase chain reaction, "target" generally
refers to the region of nucleic acid bounded by the primers used
for polymerase chain reaction. Thus, the "target" is sought to be
sorted out from other nucleic acid sequences that may be present in
a sample. A "segment" is defined as a region of nucleic acid within
the target sequence. The term "sample template" refers to nucleic
acid originating from a sample that is analyzed for the presence of
a target.
As used herein, the term "locus" refers to a particular position,
e.g., of a mutation, polymorphism, or a C residue in a CpG
dinucleotide, within a defined region or segment of nucleic acid,
such as a gene or any other characterized sequence on a chromosome
or RNA molecule. A locus is not limited to any particular size or
length, and may refer to a portion of a chromosome, a gene,
functional genetic element, or a single nucleotide or basepair. As
used herein in reference to CpG sites that may be methylated, a
locus refers to the C residue in the CpG dinucleotide.
As used herein, a "collection liquid" is a liquid in which to place
a sample to preserve, stabilize, and otherwise maintain its
integrity as a representative sample of the specimen from which the
sample was taken. While not limited in the types of compositions
that find use as collection liquids, examples of collection liquids
are aqueous buffers optionally comprising a preservative and
organic solvents, such as acetonitrile.
As used herein, "a capture reagent" refers to any agent that is
capable of binding to an analyte (e.g., a target). Preferably, "a
capture reagent" refers to any agent that is capable of
specifically binding to an analyte, e.g., having a higher binding
affinity and/or specificity to the analyte than to any other
moiety. Any moiety, such as a cell, a cellular organelle, an
inorganic molecule, an organic molecule and a mixture or complex
thereof can be used as a capture reagent if it has the requisite
binding affinity and/or specificity to the analyte. The capture
reagents can be peptides, proteins, e.g., antibodies or receptors,
oligonucleotides, nucleic acids, vitamins, oligosaccharides,
carbohydrates, lipids, small molecules, or a complex thereof.
Capture reagents that comprise nucleic acids, e.g.,
oligonucleotides, may capture a nucleic acid target by
sequence-specific hybridization (e.g., through the formation of
conventional Watson-Crick basepairs), or through other binding
interactions. When a capture oligonucleotide hybridizes to a target
nucleic acid, hybridization may involve a portion of the
oligonucleotide, or the complete oligonucleotide sequence, and the
oligonucleotide may bind to a portion of or to the complete target
nucleic acid sequence.
As used herein, "PVP" refers to polyvinylpyrrolidone, which is a
water-soluble polymer made from the monomer N-vinylpyrrolidone. The
term PVP is used herein to refer to PVP in various states of
cross-linked polymerization, including preparations of PVP that may
also be known in the art as polyvinylpolypyrrolidone (PVPP).
As used herein, a "magnet" is a material or object that produces a
magnetic field. A magnet may be a permanent magnet or an
electromagnet.
The term "amplifying" or "amplification" in the context of nucleic
acids refers to the production of multiple copies of a
polynucleotide, or a portion of the polynucleotide, typically
starting from a small amount of the polynucleotide (e.g., a single
polynucleotide molecule), where the amplification products or
amplicons are generally detectable. Amplification of
polynucleotides encompasses a variety of chemical and enzymatic
processes. The generation of multiple DNA copies from one or a few
copies of a target or template DNA molecule during a polymerase
chain reaction (PCR) or a ligase chain reaction (LCR; see, e.g.,
U.S. Pat. No. 5,494,810; herein incorporated by reference in its
entirety) are forms of amplification. Additional types of
amplification include, but are not limited to, allele-specific PCR
(see, e.g., U.S. Pat. No. 5,639,611; herein incorporated by
reference in its entirety), assembly PCR (see, e.g., U.S. Pat. No.
5,965,408; herein incorporated by reference in its entirety),
helicase-dependent amplification (see, e.g., U.S. Pat. No.
7,662,594; herein incorporated by reference in its entirety),
hot-start PCR (see, e.g., U.S. Pat. Nos. 5,773,258 and 5,338,671;
each herein incorporated by reference in their entireties),
intersequence-specfic PCR, inverse PCR (see, e.g., Triglia, et al
et al. (1988) Nucleic Acids Res., 16:8186; herein incorporated by
reference in its entirety), ligation-mediated PCR (see, e.g.,
Guilfoyle, R. et al et al., Nucleic Acids Research, 25:1854-1858
(1997); U.S. Pat. No. 5,508,169; each of which are herein
incorporated by reference in their entireties),
methylation-specific PCR (see, e.g., Herman, et al., (1996) PNAS
93(13) 9821-9826; herein incorporated by reference in its
entirety), miniprimer PCR, multiplex ligation-dependent probe
amplification (see, e.g., Schouten, et al., (2002) Nucleic Acids
Research 30(12): e57; herein incorporated by reference in its
entirety), multiplex PCR (see, e.g., Chamberlain, et al., (1988)
Nucleic Acids Research 16(23) 11141-11156; Ballabio, et al., (1990)
Human Genetics 84(6) 571-573; Hayden, et al., (2008) BMC Genetics
9:80; each of which are herein incorporated by reference in their
entireties), nested PCR, overlap-extension PCR (see, e.g., Higuchi,
et al., (1988) Nucleic Acids Research 16(15) 7351-7367; herein
incorporated by reference in its entirety), real time PCR (see,
e.g., Higuchi, et al., (1992) Biotechnology 10:413-417; Higuchi, et
al., (1993) Biotechnology 11:1026-1030; each of which are herein
incorporated by reference in their entireties), reverse
transcription PCR (see, e.g., Bustin, S. A. (2000) J. Molecular
Endocrinology 25:169-193; herein incorporated by reference in its
entirety), solid phase PCR, thermal asymmetric interlaced PCR, and
Touchdown PCR (see, e.g., Don, et al., Nucleic Acids Research
(1991) 19(14) 4008; Roux, K. (1994) Biotechniques 16(5) 812-814;
Hecker, et al., (1996) Biotechniques 20(3) 478-485; each of which
are herein incorporated by reference in their entireties).
Polynucleotide amplification also can be accomplished using digital
PCR (see, e.g., Kalinina, et al., Nucleic Acids Research. 25;
1999-2004, (1997); Vogelstein and Kinzler, Proc Natl Acad Sci USA.
96; 9236-41, (1999); International Patent Publication No.
WO05023091A2; US Patent Application Publication No. 20070202525;
each of which are incorporated herein by reference in their
entireties).
The term "polymerase chain reaction" ("PCR") refers to the method
of K. B. Mullis U.S. Pat. Nos. 4,683,195, 4,683,202, and 4,965,188,
that describe a method for increasing the concentration of a
segment of a target sequence in a mixture of genomic or other DNA
or RNA, without cloning or purification. This process for
amplifying the target sequence consists of introducing a large
excess of two oligonucleotide primers to the DNA mixture containing
the desired target sequence, followed by a precise sequence of
thermal cycling in the presence of a DNA polymerase. The two
primers are complementary to their respective strands of the double
stranded target sequence. To effect amplification, the mixture is
denatured and the primers then annealed to their complementary
sequences within the target molecule. Following annealing, the
primers are extended with a polymerase so as to form a new pair of
complementary strands. The steps of denaturation, primer annealing,
and polymerase extension can be repeated many times (i.e.,
denaturation, annealing and extension constitute one "cycle"; there
can be numerous "cycles") to obtain a high concentration of an
amplified segment of the desired target sequence. The length of the
amplified segment of the desired target sequence is determined by
the relative positions of the primers with respect to each other,
and therefore, this length is a controllable parameter. By virtue
of the repeating aspect of the process, the method is referred to
as the "polymerase chain reaction" ("PCR"). Because the desired
amplified segments of the target sequence become the predominant
sequences (in terms of concentration) in the mixture, they are said
to be "PCR amplified" and are "PCR products" or "amplicons." Those
of skill in the art will understand the term "PCR" encompasses many
variants of the originally described method using, e.g., real time
PCR, nested PCR, reverse transcription PCR (RT-PCR), single primer
and arbitrarily primed PCR, etc.
As used herein, the term "nucleic acid detection assay" refers to
any method of determining the nucleotide composition of a nucleic
acid of interest. Nucleic acid detection assay include but are not
limited to, DNA sequencing methods, probe hybridization methods,
structure specific cleavage assays (e.g., the INVADER assay,
(Hologic, Inc.) and are described, e.g., in U.S. Pat. Nos.
5,846,717, 5,985,557, 5,994,069, 6,001,567, 6,090,543, and
6,872,816; Lyamichev et al., Nat. Biotech., 17:292 (1999), Hall et
al., PNAS, USA, 97:8272 (2000), and US 2009/0253142, each of which
is herein incorporated by reference in its entirety for all
purposes); enzyme mismatch cleavage methods (e.g., Variagenics,
U.S. Pat. Nos. 6,110,684, 5,958,692, 5,851,770, herein incorporated
by reference in their entireties); polymerase chain reaction (PCR),
described above; branched hybridization methods (e.g., Chiron, U.S.
Pat. Nos. 5,849,481, 5,710,264, 5,124,246, and 5,624,802, herein
incorporated by reference in their entireties); rolling circle
replication (e.g., U.S. Pat. Nos. 6,210,884, 6,183,960 and
6,235,502, herein incorporated by reference in their entireties);
NASBA (e.g., U.S. Pat. No. 5,409,818, herein incorporated by
reference in its entirety); molecular beacon technology (e.g., U.S.
Pat. No. 6,150,097, herein incorporated by reference in its
entirety); E-sensor technology (Motorola, U.S. Pat. Nos. 6,248,229,
6,221,583, 6,013,170, and 6,063,573, herein incorporated by
reference in their entireties); cycling probe technology (e.g.,
U.S. Pat. Nos. 5,403,711, 5,011,769, and 5,660,988, herein
incorporated by reference in their entireties); Dade Behring signal
amplification methods (e.g., U.S. Pat. Nos. 6,121,001, 6,110,677,
5,914,230, 5,882,867, and 5,792,614, herein incorporated by
reference in their entireties); ligase chain reaction (e.g.,
Baranay Proc. Natl. Acad. Sci USA 88, 189-93 (1991)); and sandwich
hybridization methods (e.g., U.S. Pat. No. 5,288,609, herein
incorporated by reference in its entirety).
In some embodiments, target nucleic acid is amplified (e.g., by
PCR) and amplified nucleic acid is detected simultaneously using an
invasive cleavage assay. Assays configured for performing a
detection assay (e.g., invasive cleavage assay) in combination with
an amplification assay are described in US Patent Publication US
20090253142 A1 (App. Ser. No. 12/404,240), incorporated herein by
reference in its entirety for all purposes. Additional
amplification plus invasive cleavage detection configurations,
termed the QuARTS method, are described in U.S. patent application
Ser. Nos. 12/946,737; 12/946,745; and 12/946,752, incorporated
herein by reference in their entireties for all purposes.
The term "invasive cleavage structure" as used herein refers to a
cleavage structure comprising i) a target nucleic acid, ii) an
upstream nucleic acid (e.g., an INVADER oligonucleotide), and iii)
a downstream nucleic acid (e.g., a probe), where the upstream and
downstream nucleic acids anneal to contiguous regions of the target
nucleic acid, and where an overlap forms between the a 3' portion
of the upstream nucleic acid and duplex formed between the
downstream nucleic acid and the target nucleic acid. An overlap
occurs where one or more bases from the upstream and downstream
nucleic acids occupy the same position with respect to a target
nucleic acid base, whether or not the overlapping base(s) of the
upstream nucleic acid are complementary with the target nucleic
acid, and whether or not those bases are natural bases or
non-natural bases. In some embodiments, the 3' portion of the
upstream nucleic acid that overlaps with the downstream duplex is a
non-base chemical moiety such as an aromatic ring structure, e.g.,
as disclosed, for example, in U.S. Pat. No. 6,090,543, incorporated
herein by reference in its entirety. In some embodiments, one or
more of the nucleic acids may be attached to each other, e.g.,
through a covalent linkage such as nucleic acid stem-loop, or
through a non-nucleic acid chemical linkage (e.g., a multi-carbon
chain).
As used herein, the terms "complementary" or "complementarity" used
in reference to polynucleotides (i.e., a sequence of nucleotides)
refers to polynucleotides related by the base-pairing rules. For
example, the sequence "5'-A-G-T-3'," is complementary to the
sequence "3'-T-C-A-5'." Complementarity may be "partial," in which
only some of the nucleic acids' bases are matched according to the
base pairing rules. Or, there may be "complete" or "total"
complementarity between the nucleic acids. The degree of
complementarity between nucleic acid strands has significant
effects on the efficiency and strength of hybridization between
nucleic acid strands. This is of particular importance in
amplification reactions, as well as detection methods that depend
upon binding between nucleic acids.
As used herein, the term "primer" refers to an oligonucleotide,
whether occurring naturally, as in a purified restriction digest,
or produced synthetically, that is capable of acting as a point of
initiation of synthesis when placed under conditions in which
synthesis of a primer extension product that is complementary to a
nucleic acid strand is induced (e.g., in the presence of
nucleotides and an inducing agent such as a biocatalyst (e.g., a
DNA polymerase or the like). The primer is typically single
stranded for maximum efficiency in amplification, but may
alternatively be partially or completely double stranded. The
portion of the primer that hybridizes to a template nucleic acid is
sufficiently long to prime the synthesis of extension products in
the presence of the inducing agent. The exact lengths of the
primers will depend on many factors, including temperature, source
of primer and the use of the method. Primers may comprise labels,
tags, capture moieties, etc.
As used herein, the term "nucleic acid molecule" refers to any
nucleic acid containing molecule, including but not limited to, DNA
or RNA. The term encompasses sequences that include any of the
known base analogs of DNA and RNA including, but not limited to, 4
acetylcytosine, 8-hydroxy-N6-methyladenosine, aziridinylcytosine,
pseudoisocytosine, 5-(carboxyhydroxyl-methyl) uracil,
5-fluorouracil, 5-bromouracil,
5-carboxymethylaminomethyl-2-thiouracil,
5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,
N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,
1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine,
2-methyladenine, 2-methylguanine, 3-methyl-cytosine,
5-methylcytosine, N6-methyladenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxy-amino-methyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarbonylmethyluracil,
5-methoxyuracil, 2-methylthio-N-isopentenyladenine,
uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
oxybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
N-uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid,
pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.
As used herein, the term "nucleobase" is synonymous with other
terms in use in the art including "nucleotide," "deoxynucleotide,"
"nucleotide residue," "deoxynucleotide residue," "nucleotide
triphosphate (NTP)," or deoxynucleotide triphosphate (dNTP).
An "oligonucleotide" refers to a nucleic acid that includes at
least two nucleic acid monomer units (e.g., nucleotides), typically
more than three monomer units, and more typically greater than ten
monomer units. The exact size of an oligonucleotide generally
depends on various factors, including the ultimate function or use
of the oligonucleotide. To further illustrate, oligonucleotides are
typically less than 200 residues long (e.g., between 15 and 100),
however, as used herein, the term is also intended to encompass
longer polynucleotide chains. Oligonucleotides are often referred
to by their length. For example a 24 residue oligonucleotide is
referred to as a "24-mer". Typically, the nucleoside monomers are
linked by phosphodiester bonds or analogs thereof, including
phosphorothioate, phosphorodithioate, phosphoroselenoate,
phosphorodiselenoate, phosphoroanilothioate, phosphoranilidate,
phosphoramidate, and the like, including associated counterions,
e.g., H.sup.+, NH.sub.4.sup.+, Na.sup.+, and the like, if such
counterions are present. Further, oligonucleotides are typically
single-stranded. Oligonucleotides are optionally prepared by any
suitable method, including, but not limited to, isolation of an
existing or natural sequence, DNA replication or amplification,
reverse transcription, cloning and restriction digestion of
appropriate sequences, or direct chemical synthesis by a method
such as the phosphotriester method of Narang et al. (1979) Meth
Enzymol. 68: 90-99; the phosphodiester method of Brown et al.
(1979) Meth Enzymol. 68: 109-151; the diethylphosphoramidite method
of Beaucage et al. (1981) Tetrahedron Lett. 22: 1859-1862; the
triester method of Matteucci et al. (1981) J Am Chem Soc.
103:3185-3191; automated synthesis methods; or the solid support
method of U.S. Pat. No. 4,458,066, entitled "PROCESS FOR PREPARING
POLYNUCLEOTIDES," issued Jul. 3, 1984 to Caruthers et al., or other
methods known to those skilled in the art. All of these references
are incorporated by reference.
A "sequence" of a biopolymer refers to the order and identity of
monomer units (e.g., nucleotides, amino acids, etc.) in the
biopolymer. The sequence (e.g., base sequence) of a nucleic acid is
typically read in the 5' to 3' direction.
The term "wild-type" refers to a gene or gene product that has the
characteristics of that gene or gene product when isolated from a
naturally occurring source. A wild-type gene is that which is most
frequently observed in a population and is thus arbitrarily
designed the "normal" or "wild-type" form of the gene. In contrast,
the terms "modified," "mutant," and "variant" refer to a gene or
gene product that displays modifications in sequence and or
functional properties (i.e., altered characteristics) when compared
to the wild-type gene or gene product. It is noted that naturally
occurring mutants can be isolated; these are identified by the fact
that they have altered characteristics when compared to the
wild-type gene or gene product.
As used herein, the term "gene" refers to a nucleic acid (e.g.,
DNA) sequence that comprises coding sequences necessary for the
production of a polypeptide, precursor, or RNA (e.g., rRNA, tRNA).
The polypeptide can be encoded by a full length coding sequence or
by any portion of the coding sequence so long as the desired
activity or functional properties (e.g., enzymatic activity, ligand
binding, signal transduction, immunogenicity, etc.) of the
full-length or fragment polypeptide are retained. The term also
encompasses the coding region of a structural gene and the
sequences located adjacent to the coding region on both the 5' and
3' ends for a distance of about 1 kb or more on either end such
that the gene corresponds to the length of the full-length mRNA.
Sequences located 5' of the coding region and present on the mRNA
are referred to as 5' non-translated sequences. Sequences located
3' or downstream of the coding region and present on the mRNA are
referred to as 3' non-translated sequences. The term "gene"
encompasses both cDNA and genomic forms of a gene. A genomic form
or clone of a gene contains the coding region interrupted with
non-coding sequences termed "introns" or "intervening regions" or
"intervening sequences." Introns are segments of a gene that are
transcribed into nuclear RNA (e g., hnRNA); introns may contain
regulatory elements (e.g., enhancers). Introns are removed or
"spliced out" from the nuclear or primary transcript; introns
therefore are absent in the messenger RNA (mRNA) transcript. The
mRNA functions during translation to specify the sequence or order
of amino acids in a nascent polypeptide.
In addition to containing introns, genomic forms of a gene may also
include sequences located on both the 5' and 3' end of the
sequences that are present on the RNA transcript. These sequences
are referred to as "flanking" sequences or regions (these flanking
sequences are located 5' or 3' to the non-translated sequences
present on the mRNA transcript). The 5' flanking region may contain
regulatory sequences such as promoters and enhancers that control
or influence the transcription of the gene. The 3' flanking region
may contain sequences that direct the termination of transcription,
post-transcriptional cleavage and polyadenylation.
As used herein, the term "kit" refers to any delivery system for
delivering materials. In the context of nucleic acid purification
systems and reaction assays, such delivery systems include systems
that allow for the storage, transport, or delivery of reagents and
devices (e.g., inhibitor adsorbants, particles, denaturants,
oligonucleotides, spin filters etc. in the appropriate containers)
and/or supporting materials (e.g., buffers, written instructions
for performing a procedure, etc.) from one location to another. For
example, kits include one or more enclosures (e.g., boxes)
containing the relevant reaction reagents and/or supporting
materials. As used herein, the term "fragmented kit" refers to a
delivery system comprising two or more separate containers that
each contains a subportion of the total kit components. The
containers may be delivered to the intended recipient together or
separately. For example, a first container may contain an materials
for sample collection and a buffer, while a second container
contains capture oligonucleotides and denaturant. The term
"fragmented kit" is intended to encompass kits containing Analyte
specific reagents (ASR's) regulated under section 520(e) of the
Federal Food, Drug, and Cosmetic Act, but are not limited thereto.
Indeed, any delivery system comprising two or more separate
containers that each contains a subportion of the total kit
components are included in the term "fragmented kit." In contrast,
a "combined kit" refers to a delivery system containing all of the
components of a reaction assay in a single container (e.g., in a
single box housing each of the desired components). The term "kit"
includes both fragmented and combined kits.
The term "system" as used herein refers to a collection of articles
for use for a particular purpose. In some embodiments, the articles
comprise instructions for use, as information supplied on e.g., an
article, on paper, or on recordable media (e.g., diskette, CD,
flash drive, etc.). In some embodiments, instructions direct a user
to an online location, e.g., a website.
As used herein, the term "information" refers to any collection of
facts or data. In reference to information stored or processed
using a computer system(s), including but not limited to internets,
the term refers to any data stored in any format (e.g., analog,
digital, optical, etc.). As used herein, the term "information
related to a subject" refers to facts or data pertaining to a
subject (e.g., a human, plant, or animal). The term "genomic
information" refers to information pertaining to a genome
including, but not limited to, nucleic acid sequences, genes,
percentage methylation, allele frequencies, RNA expression levels,
protein expression, phenotypes correlating to genotypes, etc.
"Allele frequency information" refers to facts or data pertaining
to allele frequencies, including, but not limited to, allele
identities, statistical correlations between the presence of an
allele and a characteristic of a subject (e.g., a human subject),
the presence or absence of an allele in an individual or
population, the percentage likelihood of an allele being present in
an individual having one or more particular characteristics,
etc.
EMBODIMENTS OF THE TECHNOLOGY
Although the disclosure herein refers to certain illustrated
embodiments, it is to be understood that these embodiments are
presented by way of example and not by way of limitation.
1. Methods Generally
Provided herein are methods for isolating DNA, for example, from a
stool sample. As summarized in FIG. 1, the process comprises
homogenizing a sample (e.g., a stool sample) in a suitable buffer
and preparing a supernatant from the homogenate. The supernatant is
treated with a composition (e.g., a cross-linked
polyvinylpyrrolidone (PVP) such as polyvinylpolypyrrolidone (PVPP))
to remove inhibitors and produce a clarified supernatant. DNA in
the clarified supernatant is denatured, e.g., by adding guanidine
thiocyanate (GTC) and/or by heating the sample. Then, a target
capture reagent, e.g., a magnetic bead to which is linked an
oligonucleotide complementary to the target, is added and the
solution is incubated under conditions (e.g., ambient temperature
for an hour) that promote the association (e.g., by hybridization)
of the target with the capture reagent to produce a target:capture
reagent complex. After isolating and removing the target:capture
reagent complex (e.g., by application of a magnetic field), the
resulting solution is heated again to denature the remaining DNA in
the clarified supernatant and another target capture reagent can be
added to isolate another target. The process can be repeated, e.g.,
at least four times, to isolate as many targets as are required for
the assay (e.g., a sequential or serial extraction). The isolated
target:capture reagent complexes from each capture and isolation
step are washed and the target DNAs are eluted using a small volume
of buffer suitable for downstream analysis.
2. Inhibitor Removal
The sample may be a sample of material that contains impurities
that break down nucleic acids or inhibit enzymatic reactions. In
particular, such impurities inhibit the catalytic activity of
enzymes that interact with nucleic acids, e.g., nucleases such as
restriction endonucleases, reverse transcriptases, nucleic acid
polymerases, ligases, etc., particularly enzymes that are used for
polymerase chain reaction (PCR), LCR (ligase chain reaction), TMA
(transcription-mediated amplification), NASBA (nucleic acid base
specific amplification), 3SR (self-sustained sequence replication),
and the like.
2.1 PVP
In some embodiments, inhibitors in a sample are removed by
treatment with polyvinylpyrrolidone (see also, e.g., U.S. Pat.
Appl. Ser. No. 61/485,338, which is incorporated herein by
reference). Polyvinylpyrrolidone (PVP) is a water-soluble polymer
made from the monomer N-vinylpyrrolidone (see FIG. 2).
Polyvinylpolypyrrolidone (PVPP) is a highly cross-linked
modification of PVP. The extent of cross-linking varies and there
is no defined threshold establishing a division between PVP and
PVPP. Accordingly, the term PVP is used herein to refer to PVP in
various states of cross-linked polymerization, including
preparations of PVP that may also be known in the art as PVPP. An
important property, however, is that as the extent of cross-linking
is increased, the polymer becomes increasingly insoluble in water.
The cross-linked forms absorb water, which causes the polymer to
swell. The synthesis and physical properties of PVP and PVPP are
well-known in the art (e.g., see Haaf, Sanner, & Straub.
Polymers of N-vinylpyrrolidone: synthesis, characterization, and
uses. Polymer J. 17(1): 143 (1985)).
PVP has been used in many technical applications including use as a
blood plasma expander; as a binder in many pharmaceutical tablets;
as an adhesive in glue stick and hot melts; as an additive for
batteries, ceramics, fiberglass, inks, inkjet paper and in the
chemical-mechanical planarization process; as an emulsifier and
disintegrant for solution polymerization; as photoresist; for
production of membranes, such as dialysis and water purification
filters; as a thickening agent in tooth whitening gels, etc.
PVP has also found use in binding impurities and removing them from
solutions, particularly in wine-making and beer-making to remove
polyphenols (see, e.g., Redmanji, Gopal, & Mola. A novel
stabilization of beer with Polyclar Brewbrite. MBAA TQ 39(1): 24
(2002)). The use of soluble and insoluble forms of PVP has been
described in relation to processing biological samples, for
example, as a means to neutralize phenols (see, e.g., U.S. Pat. No.
7,005,266; Shames, et al. Identification of widespread Helicobacter
hepaticus infection in feces in commercial mouse colonies by
culture and PCR assay. J. Clin. Microbiol. 33(11): 2968 (1995);
Morgan et al. Comparison of PCR and microscopy for detection of
Cryptosporidium parvum in human fecal specimens: Clinical trial. J.
Clin. Microbiol. 36(4): 995 (1998)).
The PVP is provided in forms that allow its introduction into a
sample that is to be processed, e.g., as a powder, slurry,
suspension, in granules, and the like. In some embodiments of the
technology provided herein, the PVP is provided premeasured in a
ready-to-use form. For example, in some embodiments, the PVP is
pressed into a tablet comprising the mass of PVP appropriate for
treating a sample. Different sizes and shapes of tablets are
provided for different volumes and types of samples. Inert binders,
fillers, and other compositions may be added to the tablets to
provide physical, thermal, chemical, and biological stability, or
to provide other desired characteristics such as improved
dispersion within the sample or controlled-release.
Both the degree of cross-linking and the size of the PVP particles
are parameters affecting the downstream assay of the resulting
nucleic acid preparations. For example, soluble PVP has been found
to inhibit some downstream assays. Accordingly, the method benefits
from using a PVP that is sufficiently insoluble (e.g., sufficiently
cross-linked) to allow adequate removal of the PVP by downstream
processing steps (e.g., centrifugation and/or spin filtration). In
addition, when the cross-linked PVP particles are too small they
pack too tightly in the spin column and restrict the effluent flow
of the sample into the spin column collection space. For example,
experiments performed during the development of some embodiments of
the present technology demonstrated that a PVP having an average
particle size of 100-130 micrometers produced satisfactory results
while a PVP having an average particle size of 30-50 micrometers
restricted flow and filtration. Further experimentation may
indicate that other sizes and solubilities may be appropriate for
embodiments of the method.
2.2 Spin Filter
The technology provided herein encompasses use of a spin filter,
for example, as provided in U.S. Pat. Appl. Ser. No. 61/485,214 to
filter PVP-treated samples treated to remove inhibitors bound to
the PVP. As discussed above, during the development of the PVP
treatment method, experiments demonstrated that conventional spin
columns having a filter frit in the bottom end clogged under some
conditions. Accordingly, some embodiments of the technology
comprise using a clog-resistant spin filter. FIGS. 3-8 depict
various configurations of a clog-resistant spin filter in assembled
and exploded views and associated with a collection tube. The
clog-resistant filter is designed to allow the sample to be
filtered through the body walls if the bottom end becomes clogged
with residue from the sample.
Spin filters appropriate for use with the technology provided
herein are generally made from a material is inert with respect to
the sample--that is, the material does not react with or otherwise
contaminate or modify the sample, other than filtering it, in a way
that affects a subsequent assay (e.g., causes degradation of the
sample, causes its decomposition, or the like). An example of such
a material is polyethylene. Other suitable materials are, e.g.,
nylon, cellulose-acetate, polytetrafluoroethylene (PTFE, also known
as Teflon), polyvinylidene fluoride (PVDF), polyester, and
polyethersulfone. Operating pressure, the chemical and physical
characteristics of the composition to be filtered, the size of the
entity to remove from the sample, and the mechanical properties of
the material (e.g., capability to withstand centrifugation at the
speed required for the filtering application) are factors that are
considered when selecting an appropriate spin filter.
Filters are manufactured to have various pore sizes appropriate for
different filtering applications. For example, a filter with pore
size of 0.2 micrometers is typically acknowledged to remove most
bacteria while smaller pore sizes are required to remove viruses
and bacterial spores. For removing larger particulates, a larger
pore size is adequate. For example, while one aspect of the
technology provided herein uses a spin filter having a
20-micrometer pore size, other pore sizes that find use in
filtration applications are 0.22, 0.45, 10, 20, 30, and 45
micrometers. Accordingly, larger and smaller pore sizes are
contemplated, as well as pore sizes intermediate within the
intervals delimited by these particular values. For some filtration
applications the filter is characterized by the average molecular
weight of the molecules that are retained by the filter. For
example, a filter with a 5,000 Da molecular weight cutoff (MWCO) is
designed to retain molecules and complexes having at least a
molecular weight of approximately 5,000 Da. Filters can provide
MWCOs of 10,000 Da; 30,000 Da; 50,000 Da; 100,000 Da, and other
limits required for the filtration task. Operating pressure and the
size of the entity to remove from the sample are factors to
consider when choosing a pore size or cutoff value.
3. Nucleic Acid Capture
The target nucleic acids are captured using a sequence-specific
target capture reagent, e.g., a magnetic bead to which is linked an
oligonucleotide complementary to the target. After adding the
capture reagent, the solution is incubated under conditions that
promote the association (e.g., by hybridization) of the target with
the capture reagent to produce a target:capture reagent complex.
After isolating and removing the target:capture reagent complex
(e.g., by application of a magnetic field), the resulting solution
is heated again to denature the remaining DNA in the clarified
supernatant and another target capture reagent can be added to
isolate another target (e.g., by hybridization and application of a
magnetic field). The process can be repeated, e.g., at least four
times, to isolate as many targets as are required for the assay
(e.g., a sequential or serial isolation process as described, e.g.,
by U.S. Pat. Appl. Ser. No. 61/485,386, which is incorporated
herein by reference). Also, more than one target can be isolated in
a capture step by using a capture reagent comprising multiple
capture sequences.
3.1 Capture Reagents
In one aspect, the methods provided herein relate to the use of
capture reagents. Such reagents are molecules, moieties,
substances, or compositions that preferentially (e.g., specifically
and selectively) interact with a particular target sought to be
isolated and purified. Any capture reagent having desired binding
affinity and/or specificity to the analyte target is used in the
present technology. For example, in some embodiments the capture
reagent is a macromolecule such as a peptide, a protein (e.g., an
antibody or receptor), an oligonucleotide, a nucleic acid, (e.g.,
nucleic acids capable of hybridizing with the target nucleic
acids), a vitamin, an oligosaccharide, a carbohydrate, a lipid, or
a small molecule, or a complex thereof. As illustrative and
non-limiting examples, an avidin target capture reagent may be used
to isolate and purify targets comprising a biotin moiety, an
antibody may be used to isolate and purify targets comprising the
appropriate antigen or epitope, and an oligonucleotide may be used
to isolate and purify a complementary oligonucleotide (e.g., a
poly-dT oligonucleotide may be used to isolate and purify targets
comprising a poly-A tail).
Any nucleic acids, including single-, double-, and triple-stranded
nucleic acids, that are capable of binding, or specifically
binding, to the target are used as the capture reagent in the
present device. Examples of such nucleic acids include DNA, such as
A-, B- or Z-form DNA, and RNA such as mRNA, tRNA and rRNA,
aptamers, peptide nucleic acids, and other modifications to the
sugar, phosphate, or nucleoside base. Thus, there are many
strategies for capturing a target and accordingly many types of
capture reagents are known to those in the art. While not limited
in the means by which a target nucleic acid can be captured,
embodiments of the technology provided herein comprise using an
oligonucleotide that is complementary to the target and that thus
captures the target by specifically and selectively hybridizing to
the target nucleic acid.
In addition, target capture reagents comprise a functionality to
localize, concentrate, aggregate, etc. the capture reagent and thus
provide a way to isolate and purify the target when captured (e.g.,
bound, hybridized, etc.) to the capture reagent, e.g., when a
target:capture reagent complex is formed. For example, in some
embodiments the portion of the target capture reagent that
interacts with the target (e.g., the oligonucleotide) is linked to
a solid support (e.g., a bead, surface, resin, column) that allows
manipulation by the user on a macroscopic scale. Often, the solid
support allows the use of a mechanical means to isolate and purify
the target:capture reagent complex from a heterogeneous solution.
For example, when linked to a bead, separation is achieved by
removing the bead from the heterogeneous solution, e.g., by
physical movement. In embodiments in which the bead is magnetic or
paramagnetic, a magnetic field is used to achieve physical
separation of the capture reagent (and thus the target) from the
heterogeneous solution. Magnetic beads used to isolate targets are
described in the art, e.g., as described in U.S. Pat. No. 5,648,124
and European Pat. Appl. No. 87309308, incorporated herein by
reference in their entireties for all purposes.
In some embodiments, the component of the capture reagent that
interacts with the target (e.g., an oligonucleotide) is attached
covalently to the component of the capture reagent that provides
for the localization, concentration, and/or aggregation (e.g., the
magnetic bead) of the target:capture reagent complex. Exemplary
embodiments of such covalently-linked capture reagents are provided
by Stone, et al. ("Detection of rRNA from four respiratory
pathogens using an automated Q.beta. replicase assay", Molecular
and Cellular Probes 10: 359-370 (1996)), which is incorporated
herein by reference in its entirety for all purposes. These
covalently-linked capture reagents find use in the sequential
isolation of multiple specific targets from the same sample
preparation. Moreover, these capture reagents provide for the
isolation of DNA targets without many of the problems that are
associated with other methods. For example, the use of a
conventional streptavidin bead to capture a biotinylated target is
problematic for processing samples that comprise large amounts of
free biotin (e.g., a stool sample) because the free biotin
interferes with isolation of the target.
3.2 Magnetic Particle Localizer
The target:capture reagent complexes are captured using a magnetic
particle localizer. However, sample viscosity can have a profound
effect on localization efficiency due to the viscous drag affecting
the magnetic microparticles. Stool samples have viscosities ranging
from 20 centipoise to 40 centipoise, whereas, for reference, water
at 20.degree. C. has a viscosity of approximately 1 centipoise and
honey at 20.degree. C. has a viscosity of approximately 3,000
centipoise. Thus, for some applications, stronger magnetic fields
may be preferred in order to provide for a more efficient
isolation.
It has been found that particularly efficient isolations are
obtained using magnetic devices having particular arrangements of
magnets. For example, one particularly effective arrangement
provides two sets of magnets circularly arranged in parallel planar
layers around the sample, with the magnets in one layer oriented
all with their north poles toward the sample and the magnets in the
other layer are all oriented with their south poles toward the
sample (i.e., the "N-S" configuration, as opposed to other
orientations such as the "N-N" orientation in which all north poles
or all south poles in both layers are oriented toward the sample).
An example of such a device is provided by Light and Miller, U.S.
patent application Ser. No. 13/089,116 ("Magnetic Microparticle
Localization Device"), which is incorporated herein in its entirety
for all purposes. In some configurations, the magnets of the device
are arranged around a hole into which a sample tube (e.g., a 50
milliliter conical tube) is placed, such that they produce a
magnetic flux in the sample. The magnetic flux effects the movement
of the magnetic particles in the solution such that they are
aggregated, concentrated, and/or isolated in an area of the sample
tube that facilitates removal of the recovery of the target DNA
(Light, supra).
Such devices have shown to be particularly effective for the
localization of magnetic particles in large, viscous samples (e.g.,
stool samples) and thus are useful for the isolation of DNA from
such samples (Light, supra). For example, FIGS. 11A and 11B show
the effect of sample viscosity on the clearance of magnetic beads
from solutions of 1 or 25 centipoise viscosity using conventional
magnetic technology (11A) or the magnetic localization technology
of Light and Miller (11B) (Light, supra). In the graphs shown, a
decrease in absorbance indicates a decreased concentration of
microparticles suspended in solution. The data collected for the 25
centipoise solutions are shown with squares (.box-solid.) and data
collected for the 1 centipoise solution are shown with diamonds
(.diamond-solid.). These graphs show that the increase in viscosity
slows the separation dramatically when conventional technology is
used, while the Light and Miller magnetic particle localization
device clears the more viscous solution with only a modest
reduction in speed.
The chemistries and processes described above, when used in
combination, provide a system for the isolation of nucleic acids
from complex and inhibitory samples, such as stool samples, that is
significantly faster than previously used methods. Moreover, the
system produces nucleic acid preparations that are substantially
more free of inhibitory substances and results in a higher yield of
target nucleic acid for, e.g., diagnostic testing. Further,
embodiments of this system are readily integrated into the
laboratory workflow for efficient sample processing for use with
any downstream analysis or detection technology. A comparison of
the workflow, timeline, and process yields of an embodiment of the
instant system and an exemplary conventional system is shown in
FIG. 14.
4. Kits
It is contemplated that embodiments of the technology are provided
in the form of a kit. The kits comprise embodiments of the
compositions, devices, apparatuses, etc. described herein, and
instructions for use of the kit. Such instructions describe
appropriate methods for preparing an analyte from a sample, e.g.,
for collecting a sample and preparing a nucleic acid from the
sample. Individual components of the kit are packaged in
appropriate containers and packaging (e.g., vials, boxes, blister
packs, ampules, jars, bottles, tubes, and the like) and the
components are packaged together in an appropriate container (e.g.,
a box or boxes) for convenient storage, shipping, and/or use by the
user of the kit. It is understood that liquid components (e.g., a
buffer) may be provided in a lyophilized form to be reconstituted
by the user. Kits may include a control or reference for assessing,
validating, and/or assuring the performance of the kit. For
example, a kit for assaying the amount of a nucleic acid present in
a sample may include a control comprising a known concentration of
the same or another nucleic acid for comparison and, in some
embodiments, a detection reagent (e.g., a primer) specific for the
control nucleic acid. The kits are appropriate for use in a
clinical setting and, in some embodiments, for use in a user's
home. The components of a kit, in some embodiments, provide the
functionalities of a system for preparing a nucleic acid solution
from a sample. In some embodiments, certain components of the
system are provided by the user.
EXAMPLES
Example 1
During the development of embodiments of the technology provided
herein, it was demonstrated that PVP (e.g., PVPP) removes PCR
inhibitors from a stool sample (see FIG. 9). Volumes of 20
milliliters were taken from the supernatants of two different stool
supernatant samples. For each stool sample, one aliquot was treated
with PVP and the other was left untreated. Otherwise, the samples
were processed identically to capture two different nucleic acid
targets (FIG. 9, Gene A and Gene V). After capture and final
elution, the recoveries of the two targets were monitored by a SYBR
Green quantitative PCR (qPCR) assay using 1 microliter of eluate in
a 25 microliter reaction volume. For both targets from both stool
supernatants, aliquots treated with PVP were amplified whereas the
untreated aliquots failed to produce any qPCR signal. These results
demonstrate the necessity and efficacy of PVP as an
inhibitor-removal treatment when extracting DNA from stool samples
for assay by a quantitative PCR assay.
Example 2
During the development of embodiments of the technology provided
herein, data were collected demonstrating that spin filtering
improves the removal of PCR inhibitors. The experiment compared PVP
(e.g., PVPP) of different sizes for the ability to remove PCR
inhibitors from stool supernatant samples. Two commercially
available PVP compositions were compared: Polyclar.RTM. 10 and
Polyplasdone.RTM. XL, which are composed of PVP particles having an
average diameter of 30-50 micrometers and 100-130 micrometers,
respectively. Inhibitor removal by the two PVP compositions was
assessed by qPCR in which 1 microliter or 5 microliters of the
isolated DNA eluates were used in a 25-microliter reaction volume.
First, both types of PVP were separated from the stool supernatant
by pelleting (centrifugation). For both PVP types, samples showed
equal recovery and amplification curve shape when 1 microliter of
eluted DNA was added to the qPCR. However, using 5 microliters of
eluate failed to produce any qPCR signal, indicating that PCR
inhibitors remained in the sample (see FIGS. 10A and 10B).
Next, spin column filtration was tried as an alternative method to
separate the PVP from the stool supernatant. The smaller particle
size PVP could not be processed in this manner as the PVP
apparently packed down so tightly in the spin column that the
liquid stool supernatant could not pass through. However, the
larger particle size PVP did not have this same problem and the
sample preparation could easily be spin filtered. The spin column
contained a polyethylene frit (20-micrometer nominal pore size) to
collect the PVP. When separating the large particle PVP from the
stool supernatant via spin column filtration equipped with a
polyethylene frit, the eluate volume in the qPCR could be increased
to 5 microliters or 6 microliters without obvious inhibition (see
FIGS. 10C and 10D). As shown in Table 1, when using 5 or 6
microliters of eluate, the calculated strand number was
approximately five or six times the calculated strand number when
using 1 microliter of eluate. These results demonstrate the
benefits of PVP treatment plus spin column filtration for removal
of PCR inhibitors from stool samples.
TABLE-US-00001 TABLE 1 Treatment Volume Strands % Expected PVPP
30-50 1 .mu.L 950 No spin filter 5 .mu.L No Signal 0 (complete
inhibition) PVPP 100-130 1 .mu.L 907 No spin filter 5 .mu.L No
Signal 0 (complete inhibition) PVPP 100-130 1 .mu.L 1136 With spin
filter 5 .mu.L 6751 119 PVPP 100-130 1 .mu.L 3110 With spin filter
6 .mu.L 18600 99.68
Example 3
During the development of embodiments of the technology provided
herein, experiments were performed to compare the localization
efficiencies of the conventional technology (e.g., a Promega PolyA
Tract backed with a 1-inch outer diameter.times.one-eighth-inch
thick N52 neodymium magnet) and the magnetic microparticle
localizing device of Light and Miller (grade N52 neodymium magnets
in the S-N configuration) for samples of low (i.e., 1 centipoise)
and high (i.e., 25 centipoise) viscosities.
Test solutions of the appropriate viscosity (e.g., 1 or 25
centipoise) were placed in a conventional device or an embodiment
of the technology provided herein for testing. Samples were exposed
to the magnetic field, the liquid was aspirated at the time
intervals indicated for each sample, and the particles remaining in
suspension were quantified by spectrometry. A decrease in
absorbance indicates a decreased concentration of microparticles
suspended in solution (i.e., more particles localized and removed
from suspension by magnetic separation). Results for the
conventional technology are provided below in FIG. 11A. Results for
the magnetic microparticle localization device are provided in FIG.
11B. In FIGS. 11A and B, data collected for the 25 centipoise
solution are shown with squares (.box-solid.) and data collected
for the 1 centipoise solution are shown with diamonds
(.diamond-solid.).
Example 4
During the development of embodiments of the technology provided
herein, it was demonstrated that the majority of the DNA for a
given target is depleted from a stool supernatant in a single
extraction. The extraction was performed according to the flow
chart shown in FIG. 1. After final elution, the recoveries of the
two targets (Gene A and Gene V) from extractions 1 and 2 were
monitored by SYBR Green qPCR assays using 1 microliter of eluate in
a 25-microliter volume reaction. For both targets, extraction 1
yielded good recovery of target, whereas the eluate from extraction
2 failed to produce any qPCR signal for either target (FIG.
12).
Example 5
During the development of embodiments of the technology provided
herein, it was demonstrated that DNA extraction can be performed
repeatedly on a single sample through a minimum of four cycles of
denaturation/hybridization without compromising the integrity of
the human DNA in the stool supernatant. In this example, four
targets (Genes A, F, V, and W) were captured from the sample and
the order of their capture was varied. After elution, the recovery
of each target was monitored by SYBR Green qPCR. In FIG. 13, plots
show the amplification curves for each gene when it was captured
first, second, third, and fourth in the extraction sequence. The
superposition of the amplification curves demonstrates that
recoveries were approximately equal regardless of the order of
extraction. Table 3 quantifies the results from FIG. 13.
TABLE-US-00002 TABLE 3 Target Extraction Mean C.sub.p Mean
Strands/.mu.L Gene A #1 28.92 862 #2 28.89 878 #3 28.85 907 #4
28.73 984 Gene F #1 29.32 499 #2 29.36 489 #3 29.29 511 #4 29.01
614 Gene V #1 31.29 129 #2 31.01 155 #3 31.18 139 #4 30.84 177 Gene
W #1 29.17 724 #2 29.11 757 #3 28.99 819 #4 29.16 730
For all four genes, the mean C.sub.p (Crossing point--the cycle
number at which the amplification curve crosses a fixed threshold)
and strand numbers were essentially equal regardless of the order
of extraction.
Example 6
Exemplary Procedure for Serial Isolation of a Plurality of Target
Nucleic acids:
As diagrammed in FIG. 1: 1. A stool sample is homogenized, e.g.,
with a buffer, to form a stool homogenate. The homogenate treated
to partition residual solids from the fluid, e.g., by
centrifugation or filtration, to produce a "stool supernatant." 2.
Stool supernatant is treated to remove assay inhibitors (e.g., with
polyvinylpolypyrrolidone, as described in U.S. Pat. Appl. Ser. No.
61/485,338, which is incorporated herein by reference in its
entirety), producing "clarified supernatant". 3. Ten milliliters of
clarified supernatant (representing an equivalent of approximately
4 grams of stool) is mixed with guanidine thiocyanate (GTC) to a
final concentration of 2.4 M; 4. The mixture is then heated in a
90.degree. C. water bath for 10 minutes to denature the
DNA (and proteins) present in the stool. 5. Paramagnetic particles
containing covalently attached (coupled) oligonucleotides
complementary to the target sequence(s) of interest
("target-specific capture probes") are added to the sample. The
sample is then incubated (e.g., at ambient temperature, about
22-25.degree. C.) for one hour to enable hybridization of the
target DNA to the capture probes on the magnetic particles. 6. The
mixture of clarified supernatant, GTC, and particles is exposed to
a magnetic field to separate the particles (now containing target
DNA hybridized to the capture probes) from the stool
supernatant/GTC mixture, which is transferred to a new tube. See,
e.g., U.S. patent application Ser. No. 13/089,116, which is
incorporated herein by reference. 7. The paramagnetic particles are
then washed and the target DNA eluted, ready for use in detection
assays. 8. The supernatant/GTC mixture retained in step 6 is
returned to the 90.degree. C. water bath for 10 minutes to repeat
denaturation (step 4). Step 5 is then repeated by adding magnetic
particles containing capture probes complementary to different
targets DNAs, and the hybridization, particle separation and
elution steps are repeated to produce a purified sample of a second
DNA target.
The denaturation/hybridization/separation cycle (steps 4-6) can be
repeated at least four or more times to serially extract different
target DNAs from the same stool supernatant sample.
Example 7
During the development of embodiments of the technology provided
herein, the methods were tested in a clinical application. The
following provides an example of workflow using the systems and
methods of the present invention.
Study Design
This study was based on well-characterized archival stools from
multiple medical centers, including referral centers and community
medical centers in the United States and Denmark. Approval by
institutional review boards was obtained. Stools were procured from
case patients with proven colorectal cancer (CRC), cases with at
least one colorectal adenoma.gtoreq.1 centimeter, and age and sex
matched control patients without neoplasia as assessed by
colonoscopy. Patients had been recruited from both clinical and
screening settings, and some were symptomatic. Those with known
cancer syndromes or inflammatory bowel disease were excluded.
Nearly 700 samples were tested, of which 133 were adenomas.gtoreq.1
centimeter and 252 were cancer patients.
A multi-marker stool test was performed that included four
methylated genes (vimentin, NDRG4, BMP3, and TFPI2), mutant KRAS, a
reference gene beta-actin (ACTB), and hemoglobin. To evaluate test
performance, case and control stools were distributed in balanced
fashion to two different test sites; all assays were run by blinded
technicians.
Stool Collection and Storage.
Prior to colonoscopy, which served as the gold standard, whole
stools were collected in plastic buckets. A preservative buffer was
added to the stool and buffered stools were archived at -80.degree.
C. However, the timing of buffer addition, duration between
defecation and freezing, and whether or not samples were
homogenized prior to storage were not standardized and varied
across participating centers.
Marker Selection.
Candidate genes were identified that individually or in
combinations (e.g., KRAS+BMP3+NDRG4+TFPI2+vimentin+reference and/or
ACTB+hemoglobin) yielded nearly complete separation of colorectal
neoplasia from normal mucosa. Four methylated gene markers emerged
as the most discriminant--NDRG4, BMP3, vimentin, and TFPI2. Mutant
KRAS and hemoglobin detection complement methylated gene markers
detected in stool and, accordingly, were also evaluated in the
marker panel. Finally, assay of the reference gene beta-actin
(ACTB) was used to determine total human genome equivalents in
stool and, as human DNA levels in stool increase with colorectal
neoplasia, to serve as a candidate marker itself.
Stool Processing and Target Gene Capture
Promptly after thawing, buffered stools were thoroughly homogenized
and centrifuged. A 14-milliliter aliquot of stool supernatant was
then treated with polyvinylpolypyrrolidone at a concentration of 50
milligrams per milliliter. Direct capture of target gene sequences
by hybridization with oligonucleotide probes was performed on
supernatant material. Briefly, 10 milliliters of insoluble
PVP-treated supernatant was denatured in 2.4 M guanidine
isothiocyanate (Sigma, St. Louis Mo.) at 90.degree. C. for 10
minutes; 300-500 micrograms of Sera-Mag carboxylate modified beads
(ThermoFisher Scientific, Waltham Mass.) functionalized with each
oligonucleotide capture probe were subsequently added to denatured
stool supernatant and incubated at room temperature for one hour.
Sera-Mag beads were collected on a magnetic rack and washed three
times using MOPS washing buffer (10 mM MOPS; 150 mM NaCl, pH 7.5),
and then eluted in 60 microliters of nuclease free water with 20
nanograms per microliter tRNA (Sigma). In this study, four selected
methylated markers, vimentin, NDRG4, BMP3, and TFPI2, and one
reference gene ACTB, were captured together in one hybridization
reaction; the mutation marker KRAS was subsequently captured in
another hybridization reaction. The capture probes used, shown here
with their 5'-six carbon amino modified linkage (Integrated DNA
Technology, Coralville, Iowa), were as follows:
TABLE-US-00003 for vimentin: (SEQ ID NO: 1)
/5AmMC6/CTGTAGGTGCGGGTGGACGTAGTCACGTAGCTCCGGCTGGA-3'; for NDRG4:
(SEQ ID NO: 2)
/5AmMC6/TCCCTCGCGCGTGGCTTCCGCCTTCTGCGCGGCTGGGGTGCCCGGTGG-3'; for
BMP3: (SEQ ID NO: 3) /5AmMC6/GCGGGACACTCCGAAGGCGCAAGGAG-3'; for
TFPI2: (SEQ ID NO: 4) /5AmMC6/CGCCTGGAGCAGAAAGCCGCGCACCT-3'; for
ACTB: (SEQ ID NO: 5) /5AmMC6/CCTTGTCACACGAGCCAGTGTTAGTACCTACACC-3';
for KRAS: (SEQ ID NO: 6)
/5AmMC6/GGCCTGCTGAAAATGACTGAATATAAACTTGTGGTAGTTGGAGC-3' and (SEQ ID
NO: 7)
/5AmMC6/CTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATTAGC-3'
Methylation Assays.
Methylated markers were quantified by the QuARTS method, as we have
previously described (see, e.g., U.S. patent application Ser. Nos.
12/946,737; 12/946,745; and 12/946,752, incorporated herein by
reference in their entireties for all purposes). This method
combines a polymerase-based target DNA amplification process with
an invasive cleavage-based signal amplification process. We treated
45 microliters of captured DNA with bisulfite using the EZ-96 DNA
Methylation Kit (Zymo Research, Irvine Calif.) and eluted the
sample in 50 microliters of 10 mM Tris, 0.1 mM EDTA pH 8.0 with 20
nanograms per microliter tRNA (Sigma) on a 96-well PCR plate; 10
microliters of bisulfite-treated DNA was assayed with the QuARTS
method in 30-microliter reaction volumes on a 96-well PCR plate.
PCR plates were cycled in a LightCycler 480 (Roche).
Two separate triplex QuARTS assays were designed to detect the
methylated markers vimentin, NDRG4, BMP3, and TFPI2 using ACTB as a
reference gene for each. The first triplex assay contained ACTB,
vimentin, and NDRG4, and the second contained ACTB, BMP3, and
TFPI2. Each QuARTS reaction incorporated 400-600 nM primers and
detection probes, 100 nM invasive oligonucleotide, 600-700 nM each
of FAM (Hologic, Madison Wis.), Yellow (Hologic), and Quasor 670
(BioSearch Technologies, Novato Calif.) fluorescence resonance
energy transfer reporter cassettes (FRETs), 6.675 nanogram per
microliter Cleavase 2.0 (Hologic), 1 unit hot-start GoTaq DNA
polymerase (Promega, Madison Wis.), 10 mM MOPS, 7.5 mM MgCl.sub.2,
and 250 .mu.M each dNTP. QuARTS cycling conditions consisted of
95.degree. C. for 3 minutes, then 10 cycles each comprising
95.degree. C. for 20 seconds, 67.degree. C. for 30 seconds, and
70.degree. C. for 30 seconds, followed by 45 cycles each comprising
95.degree. C. for 20 seconds, 53.degree. C. for 1 minute, and
70.degree. C. for 30 seconds, and finally a 30-second hold at
40.degree. C. For each target below, the two methylation-specific
primers and probe (Integrated DNA Technology, Coralville, Iowa)
were as follows:
TABLE-US-00004 For vimentin: Primer (SEQ ID NO: 8) 5'-GGC GGT TCG
GGT ATC G-3', Primer (SEQ ID NO: 9) 5'-CGT AAT CAC GTA ACT CCG AC
T-3', Probe (SEQ ID NO: 10) 5'-GAC GCG GAG GCG AGT CGG TCG/3'C6/;
for NDRG4: Primer (SEQ ID NO: 11) 5'-CGG TTT TCG TTC GTT TTT
TCG-3', Primer (SEQ ID NO: 12) 5'-GTA ACT TCC GCC TTC TAC GC-3',
Probe (SEQ ID NO: 13) 5'-CGC CGA GGG TTC GTT TAT CG/3'C6/; for
BMP3: Primer (SEQ ID NO: 14) 5'-GTT TAA TTT TCG GTT TCG TCG TC-3',
Primer (SEQ ID NO: 15) 5'-CTC CCG ACG TCG CTA CG-3', Probe (SEQ ID
NO: 16) 5'-CGC CGA GGC GGT TTT TTG CG/3'C6/; and for TFPI2: Primer
(SEQ ID NO: 17) 5'-TCG TTG GGT AAG GCG TTC-3', Primer (SEQ ID NO:
18) 5'-AAA CGA ACA CCC GAA CCG-3', Probe (SEQ ID NO: 19) 5'-GAC GCG
GAG GCG GTT TTT TGT T/3'C6/.
The TFPI2 assay had a specific invasive oligonucleotide:
TABLE-US-00005 (SEQ ID NO: 20) 5'-GCG GGA GGA GGT GCC-3'.
Primers and probe for detecting bisulfite-treated ACTB were:
TABLE-US-00006 Primer (SEQ ID NO: 21) 5'-TTT GTT TTT TTG ATT AGG
TGT TTA AGA-3', Primer (SEQ ID NO: 22) 5'-CAC CAA CCT CAT AAC CTT
ATC-3', Probe (SEQ ID NO: 23) 5'-CCA CGG ACG ATA GTG TTG TGG/
3'C6/.
Each plate included bisulfite-treated DNA samples, standard curve
samples, positive and negative controls, and water blanks Standard
curves were made using 300 to 1000 target sequences cut from
engineered plasmids. Bisulfite-treated CpGenome universal
methylated DNA (Millipore, Billerica, Mass.) and human genomic DNA
(Merck, Germany) were used as positive and negative controls. DNA
strand number was determined by comparing the C.sub.p of the target
gene to the standard curve for the relevant assay. Percent
methylation for each marker was determined by dividing the strand
number of the methylated gene by the ACTB strand number and
multiplying by 100.
KRAS Mutation
The KRAS gene was first PCR amplified with primers flanking codons
12/13 using 10 microliters of captured KRAS DNA as template. PCR
was conducted with 1.times. LightCycler.RTM. 480 SYBR Green I
Master (Roche, Germany) and 200 nM each primer. Cycling conditions
were 95.degree. C. for 3 minutes, followed by 15 cycles each at
95.degree. C. for 20 seconds, 62.degree. C. for 30 seconds, and
72.degree. C. for 30 seconds. Primer sequences were:
TABLE-US-00007 (SEQ ID NO: 24) 5'-AGG CCT GCT GAA AAT GAC TG-3',
and (SEQ ID NO: 25) 5'-CTA TTG TTG GAT CAT ATT CG TC- 3'.
Each amplified sample was diluted 500-fold in nuclease free water.
A 10-microliter aliquot of the 500-fold sample dilutions was added
to a 96-well PCR plate with an automated liquid handler (epMotion,
Eppendorf, Hauppauge N.Y.). QuARTS assays were then used to
evaluate seven mutations at codons 12/13 of the KRAS gene. Each
mutation assay was designed as a singleplex assay. KRAS
mutation-specific forward primers and probes were:
TABLE-US-00008 for G12S mutation: Primer (SEQ ID NO: 26) 5'-CTT GTG
GTA GTT GGA GCA A-3' Probe (SEQ ID NO: 27) 5'-GCG CGT CCA GTG GCG
TAG GC/3'C6/; for G12C mutation Primer (SEQ ID NO: 28) 5'-AAA CTT
GTG GTA GTT GGA CCT T-3' Probe (SEQ ID NO: 29) 5'-GCG CGT CCT GTG
GCG TAG GC/3'C6/; for G12R mutation Primer (SEQ ID NO: 30) 5'-TAT
AAA CTT GTG GTA GTT GGA CCT C-3' Probe (SEQ ID NO: 31) 5'-GCG CGT
CCC GTG GCG TAG GC/3'C6/; for G12D mutation Primer (SEQ ID NO: 32)
5'-ACT TGT GGT AGT TGG AGC TCA-3' Probe (SEQ ID NO: 33) 5'-GCG CGT
CCA TGG CGT AGG CA/3'C6/; for Gl2V mutation Primer (SEQ ID NO: 34)
5'-ACT TGT GGT AGT TGG AGC TCT-3' Probe (SEQ ID NO: 35) 5'-GCG CGT
CCT TGG CGT AGG CA/3'C6/; for G12A mutation Primer (SEQ ID NO: 36)
5'-AAC TTG TGG TAG TTG GAG ATG C-3' Probe (SEQ ID NO: 37) 5'-GCG
CGT CCC TGG CGT AGG CA/3'C6/; for G13D mutation Primer (SEQ ID NO:
38) 5'-GGT AGT TGG AGC TGG TCA-3' Probe (SEQ ID NO: 39) 5'-GCG CGT
CCA CGT AGG CAA GA/3'C6/.
For all KRAS mutants, the reverse primer used is
TABLE-US-00009 (SEQ ID NO: 40) 5'-CTA TTG TTG GAT CAT ATT CGT
C-3'.
QuARTS cycling conditions and reagent concentrations for KRAS were
the same as those in the methylation assays. Each plate contained
standards made of engineered plasmids, positive and negative
controls, and water blanks, and was run in a LightCycler 480
(Roche). DNA strand number was determined by comparing the C.sub.p
of the target gene to the standard curve for that assay. The
concentration of each mutation marker in 50 microliters of KRAS was
calculated based on the 500-fold dilution factor and an
amplification efficiency of 1.95. This value was divided by the
ACTB concentration in the methylation assay and then multiplied by
100 to determine the percent mutation.
Hemoglobin Assay.
To quantify hemoglobin in stool, the semi-automated HemoQuant test
was performed on two buffered stool aliquots (each normalized to 16
milligrams of stool) per patient, as described in Ahlquist, et al.
("HemoQuant, a new quantitative assay for fecal hemoglobin.
Comparison with Hemoccult". Ann Intern Med 101:297-302 (1984)).
This test allowed assessment of the complementary value of fecal
hemoglobin.
Data Analysis
Using the combination of sample processing methods described
herein, comprising inhibitor removal and target capture
purification, combined with the methylation and mutation markers
described, the present study of 678 samples achieved the following
sensitivity levels: 63.8% sensitivity for adenoma detection and
85.3% sensitivity for colorectal cancer at a specificity level of
90%.
All publications and patents mentioned in the above specification
are herein incorporated by reference in their entirety for all
purposes. Various modifications and variations of the described
compositions, methods, and uses of the technology will be apparent
to those skilled in the art without departing from the scope and
spirit of the technology as described. Although the technology has
been described in connection with specific exemplary embodiments,
it should be understood that the invention as claimed should not be
unduly limited to such specific embodiments. Indeed, various
modifications of the described modes for carrying out the invention
that are obvious to those skilled in pharmacology, biochemistry,
medical science, or related fields are intended to be within the
scope of the following claims.
SEQUENCE LISTINGS
1
40141DNAArtificial SequenceSynthetic 1ctgtaggtgc gggtggacgt
agtcacgtag ctccggctgg a 41248DNAArtificial SequenceSynthetic
2tccctcgcgc gtggcttccg ccttctgcgc ggctggggtg cccggtgg
48326DNAArtificial SequenceSynthetic 3gcgggacact ccgaaggcgc aaggag
26426DNAArtificial SequenceSynthetic 4cgcctggagc agaaagccgc gcacct
26534DNAArtificial SequenceSynthetic 5ccttgtcaca cgagccagtg
ttagtaccta cacc 34644DNAArtificial SequenceSynthetic 6ggcctgctga
aaatgactga atataaactt gtggtagttg gagc 44746DNAArtificial
SequenceSynthetic 7ctctattgtt ggatcatatt cgtccacaaa atgattctga
attagc 46815DNAArtificial SequenceSynthetic 8ggcggttcgg gtatc
15921DNAArtificial SequenceSynthetic 9cgtaatcacg taactccgac t
211021DNAArtificial SequenceSynthetic 10gacgcggagg cgagtcggtc g
211121DNAArtificial SequenceSynthetic 11cggttttcgt tcgttttttc g
211220DNAArtificial SequenceSynthetic 12gtaacttccg ccttctacgc
201320DNAArtificial SequenceSynthetic 13cgccgagggt tcgtttatcg
201423DNAArtificial SequenceSynthetic 14gtttaatttt cggtttcgtc gtc
231517DNAArtificial SequenceSynthetic 15ctcccgacgt cgctacg
171620DNAArtificial SequenceSynthetic 16cgccgaggcg gttttttgcg
201718DNAArtificial SequenceSynthetic 17tcgttgggta aggcgttc
181818DNAArtificial SequenceSynthetic 18aaacgaacac ccgaaccg
181922DNAArtificial SequenceSynthetic 19gacgcggagg cggttttttg tt
222015DNAArtificial SequenceSynthetic 20gcgggaggag gtgcc
152127DNAArtificial SequenceSynthetic 21tttgtttttt tgattaggtg
tttaaga 272221DNAArtificial SequenceSynthetic 22caccaacctc
ataaccttat c 212321DNAArtificial SequenceSynthetic 23ccacggacga
tagtgttgtg g 212420DNAArtificial SequenceSynthetic 24aggcctgctg
aaaatgactg 202522DNAArtificial SequenceSynthetic 25ctattgttgg
atcatattcg tc 222619DNAArtificial SequenceSynthetic 26cttgtggtag
ttggagcaa 192720DNAArtificial SequenceSynthetic 27gcgcgtccag
tggcgtaggc 202822DNAArtificial SequenceSynthetic 28aaacttgtgg
tagttggacc tt 222920DNAArtificial SequenceSynthetic 29gcgcgtcctg
tggcgtaggc 203024DNAArtificial SequenceSynthetic 30tataaacttg
tggtagttgg acct 243120DNAArtificial SequenceSynthetic 31gcgcgtcccg
tggcgtaggc 203221DNAArtificial SequenceSynthetic 32acttgtggta
gttggagctc a 213320DNAArtificial SequenceSynthetic 33gcgcgtccat
ggcgtaggca 203421DNAArtificial SequenceSynthetic 34acttgtggta
gttggagctc t 213520DNAArtificial SequenceSynthetic 35gcgcgtcctt
ggcgtaggca 203622DNAArtificial SequenceSynthetic 36aacttgtggt
agttggagat gc 223720DNAArtificial SequenceSynthetic 37gcgcgtccct
ggcgtaggca 203818DNAArtificial SequenceSynthetic 38ggtagttgga
gctggtca 183920DNAArtificial SequenceSynthetic 39gcgcgtccac
gtaggcaaga 204022DNAArtificial SequenceSynthetic 40ctattgttgg
atcatattcg tc 22
* * * * *
References